CN111910012A - Fluorescent PCR method for detecting point mutation of clostridium difficile moxifloxacin-resistant gyrA gene - Google Patents

Abstract

The invention provides a group of polynucleotides for fluorescent PCR primers and probes and a fluorescent PCR method for detecting the gyrA gene point mutation of clostridium difficile. The method provided by the invention simplifies the detection workload, avoids obtaining gyrA gene point mutation information by amplification sequencing comparison, and shortens the detection time; the detection sensitivity of the kit to both moxifloxacin drug-resistant clostridium difficile and moxifloxacin sensitive clostridium difficile is 10¹Copying; the kit has no non-specific amplification to 6 common intestinal bacteria in clinic, and shows good specificity. The inventionThe method has stable reaction system, shows good repeatability between groups, and has higher consistency and accuracy of detection results compared with gene amplification sequencing.

Description

Fluorescent PCR method for detecting point mutation of clostridium difficile moxifloxacin-resistant gyrA gene

Technical Field

The invention discloses a group of polynucleotides and application thereof in detecting gene point mutation, belonging to the field of nucleotide application.

Background

Difficile is an important etiology of Antibiotic-associated diarrhea and is listed as the third place of urgent public Health threats by the U.S. CDC (reference: u.s. department of Health and Human Services, CDC, Antibiotic resistance diseases in the united states sites 2019). The BI/NAP1/RT027 strain caused a multiple outbreak epidemic in North America, Europe and Australia with high levels of quinolone resistance, toxin secretion and lethality (ref: Daniel A. N Engl J Med 2015; 372: 1539-48). Traditional drug-resistant strains are mainly dependent on phenotypic experimental findings of drug susceptibility tests, such as agar dilution, broth dilution, and commercial E-test strips. In recent years, with the spread of whole genome sequencing technologies, it has become possible to infer drug-resistant phenotypes based on drug-resistant gene analysis (references: David W Eyre, et al. J. Antichronob Chemother 2017; 72(7):1937-1947.N Stosser, et al. J. Antichronob Chemother 2013; 68(10): 2234-44.). According to the previous experimental results and literature reports of the applicant, point mutation of the gyrA gene (Thr → IIe) and moxifloxacin resistance of clostridium difficile are found to have high correlation. The gyrA gene, DNA gyrase subustit A, is located in the quinolone drug resistance determining region (QRDR) and is related to fluoroquinolone drug resistance. The point mutation Thr → IIe, i.e.the amino acid at position 82 is mutated from Thr to Ile (genbank accession number PRJNA479396) and the corresponding nucleotide from ACT to ATT. The judgment of sensitivity or drug resistance of clostridium difficile to moxifloxacin can be prompted by detecting the point mutation of the gene, and when the mutation is detected to be ACT, the judgment of sensitivity to moxifloxacin is prompted; when the mutation is ATT, the detection suggests that the moxifloxacin is resistant (the reference: Yuan Wu et al. mSystems,2019,4(2): e00252-18.Patrizia Spiglia. Ther Adv Infect Dis 2016; 3(1): 23-42).

However, at present, no fluorescent PCR method aiming at the gyrA gene point mutation of the clostridium difficile exists at home and abroad. Through the search of other bacteria related researches, the detection of point mutation of the gyrA gene related to quinolone drug resistance is found mainly by relying on the common PCR amplification sequencing comparison or annealing temperature change to find the point mutation (the reference document: Yi Mao et al, J.Utility medicine, 2010,26(005): 724: 726. Wang hong Ning et al, CN 104611422A). Compared with the fluorescent PCR method, the method has more operation steps and longer time consumption, and is not easy to be combined with the fluorescent PCR method for detecting the clostridium difficile.

Therefore, the invention aims to quickly detect the drug-resistant or sensitive strains of the clostridium difficile, and can be combined with the existing fluorescent PCR method for producing the toxic clostridium difficile to play a role in prompting and early warning for judging the epidemic strains of the clostridium difficile (BI/NAP1/RT 027).

Disclosure of Invention

The invention aims to provide a method which has high sensitivity, high specificity, good repeatability and high accuracy and can simultaneously detect the drug resistance or sensitivity of the clostridium difficile moxifloxacin, improve the detection capability of the drug resistance or sensitivity of the clostridium difficile moxifloxacin, and play a role in prompting and early warning the judgment of the clostridium difficile epidemic strains by combining with the toxicity detection of the clostridium difficile.

In view of the above objects, the present invention provides, in a first aspect, a set of polynucleotides for use in fluorescent PCR primers and probes, the polynucleotides comprising: an upstream primer Gyr-F with a sequence shown as SEQ ID NO.1, a downstream primer Gyr-R with a sequence shown as SEQ ID NO.2, a probe P-ATT with a sequence shown as SEQ ID NO.3 and a probe P-ACT2 with a sequence shown as SEQ ID NO. 4.

In a preferred embodiment, the probe P-ATT is labeled with a first fluorescent emitting group and a fluorescence quenching gene, and the probe P-ACT2 is labeled with a second fluorescent emitting group and a fluorescence quenching gene.

In a more preferred embodiment, the probe P-ATT is labeled at its 5 'end with a first fluorescent emitting group, the probe P-ATT is labeled at its 3' end with a fluorescence quenching gene, the probe P-ACT2 is labeled at its 5 'end with a second fluorescent emitting group, and the probe P-ACT2 is labeled at its 3' end with a fluorescence quenching gene.

More preferably, the first fluorescent group is VIC, the second fluorescent group is FAM, and the fluorescence quencher group is MGB.

In an alternative preferred embodiment, the first fluorescent emitting group is FAM, the second fluorescent emitting group is VIC, and the fluorescence quenching group is MGB.

Secondly, the present invention provides a fluorescent PCR method for detecting clostridium difficile gyrA gene point mutation for non-diagnostic purposes, the method comprising the steps of:

(1) preparing genome DNA of a sample to be detected;

(2) preparing a combination of polynucleotides as described above as amplification primers and probes;

(3) amplifying the genomic DNA in a PCR working environment in which the amplification primers are present;

(4) and (4) detecting the amplification result of the step (3).

In a preferred embodiment, the annealing temperature of the PCR working environment is 56-60 ℃.

In a more preferred embodiment, the annealing temperature of the PCR working environment is 58-59 ℃. In one embodiment of the present invention, the annealing temperature of the PCR working environment is set to 59 ℃.

In another preferred embodiment, the detection of step (4) is a fluorescent quantitative detection.

Finally, the invention provides a kit comprising a combination of polynucleotides as described above.

The invention designs specific probes and primers for point mutation of gyrA genes of drug-resistant and sensitive strains of clostridium difficile respectively, establishes a method for simultaneously detecting the two types of clostridium difficile by a single reaction through laboratory condition optimization, simplifies the detection workload, avoids obtaining point mutation information of the gyrA genes through amplification sequencing comparison, shortens the detection time, and can shorten the detection time to 1 hour compared with 48 hours required by common PCR amplification sequencing; the method of the invention is used for the moxifloxacin drug-resistant clostridium difficile and moxifloxacin sensitive difficileThe detection sensitivity of the clostridium is 10¹Copying; the method has no non-specific amplification to 6 common intestinal bacteria in clinic, and shows good specificity; and compared with the gene amplification sequencing, the detection result of the method has higher consistency and accuracy. In addition, the method has stable reaction system and shows good repeatability in groups and between groups.

Drawings

FIG. 1 is a schematic diagram showing the positions of mutation sites and primers and probes on the gyrA gene;

FIG. 2 is a graph showing the amplification at 59 ℃ for an annealing temperature;

FIG. 3 is a standard graph of a resistance to moxifloxacin clostridium difficile system;

FIG. 4 is a standard graph of a Clostridium difficile system sensitive to moxifloxacin;

FIG. 5 is a diagram of specific detection amplification;

FIG. 6.73 alignment chart of gyrA gene partial sequencing of Clostridium difficile;

FIG. 7.73 Clostridium difficile was amplified using the dual fluorescence PCR assay of the present invention.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are only illustrative and do not limit the scope of protection defined by the claims of the present invention.

Through previous clostridium difficile drug sensitivity tests and correlation analysis (GWAS) based on whole genome sequencing, point mutation (Thr → IIe) of the gyrA gene is found to have higher correlation with moxifloxacin resistance of clostridium difficile. The point mutation of the gene can be detected to prompt that the clostridium difficile is sensitive or resistant to moxifloxacin.

The invention designs specific probes and primers for point mutation of gyrA genes of drug-resistant and sensitive strains of clostridium difficile. Based on the existing drug-resistant and sensitive gyrA gene sequence (genbank accession number: PRJNA479396.), primer express3.0 is applied to design primer probes, and sequence specificity is verified in the primer blast of NCBI. Finally obtaining a pair of specific primers:

Gyr-F: TGTGCCATTCTTACCATA (SEQ ID NO.1), and

Gyr-R：AAGCCAGTTCATAGAAGA(SEQ ID NO.2)；

and a probe P-ATT of the drug-resistant point mutation ATT:

5 'VIC-TAAACAGCAATATCTCC-MGB 3' (SEQ ID NO.3), and

probe P-ACT2 for sensitive Strain ACT:

5’FAM-ATAATAAACAGCAGTATC-MGB 3’(SEQ ID NO.4)。

the total length of the gyrA gene is 2427bp, and the positions of a mutation site and a primer probe are shown by taking the sensitive strain 630 and the drug-resistant R20291 shown in figure 1 as an example: the sequence marked by single-dashed lines is a primer, the sequence marked by double-dashed lines is a probe, and gray in the probe sequence is marked as a sequence mutation region. The initial position of the Gyr-F primer on the sensitive strain 630 is 121bp, and the initial position of the drug-resistant strain R20291 is 133 bp; the initial position of the Gyr-R primer on the sensitive strain 630 is 261bp, and the initial position of the drug-resistant strain R20291 is 279 bp; the probe P-ACT2 is 239bp at the initial position of the sensitive strain 630; the initial position of the probe P-ATT in the drug-resistant strain R20291 is 253 bp.

The above sequences were synthesized by Biotechnology, Inc.

The fluorescent PCR reaction solution and fluorescent reagent required for the experiment are purchased from TaKaRa, and the instrument is 7500Fast of ABI.

Wherein, the 5 'end of the probe P-ATT is labeled with a fluorescent dye VIC, the 3' end is labeled with a fluorescence quenching group MGB, the 5 'end of the probe P-ACT2 is labeled with a fluorescent dye FAM, and the 3' end is labeled with a fluorescence quenching group MGB (as an alternative, the 5 'end of the probe P-ATT can also be labeled with a fluorescent dye FAM, and the 5' end of the probe P-ACT2 is labeled with a fluorescent dye VIC).

The detection principle of the method is as follows: allele differential Analysis (AD) using the TaqMan probe fluorescence PCR method is a multiplex (each reaction includes more than one primer and probe pair), endpoint (data collected at the end of the PCR process) analysis experiment for detecting variations in a nucleic acid sequence. Including two primer and probe pairs in each reaction, allowing for the sequencing of one target templateTwo possible variant genotypes occur at Single Nucleotide Polymorphism (SNP) sites. For each sample in an allele-discriminating (AD) assay, a unique pair of fluorescent probes, e.g., two, is used

MGB probes were used to label one SNP site. One perfectly matched the wild type (allele 1) and the other perfectly matched the mutation (allele 2). The result is determined by analytically measuring the change in the fluorescent signal associated with the probe. If some fluorescence is obviously increased, the combination of the probe for marking the fluorescence and the gene with the mutation is shown as allele 1 or 2, and the specific application is the strain type pointed by the fluorescent probe combined with the specific gene, namely sensitive strain ACT or drug-resistant strain ATT. The fluorescence quenching group MGB terminates the reaction at the end of the reaction. The fluorescence quenching group MGB (minor Groove binder) is a new probe technology introduced by ABI company in the United states in 2000, and solves the problem that the traditional probe is not complete in fluorescence quenching. The hybridization between the MGB-labeled probe and the template is more stable, so that the shorter probe can reach a higher Tm value, and the detection of single base mutation is possible because the distance between the fluorescent reporter group and the quenching group of the short probe is closer, the quenching effect is better, the fluorescent background is lower, the signal-to-noise ratio is higher, and the allele differentiation is more ideal.

Example 1 multiplex fluorescent PCR amplification System and amplification conditions

The experimental steps are as follows:

1. extracting the genomic DNA of clostridium difficile to be detected;

2. configuring a reaction system as follows;

a detection system:

3. detecting on a machine;

the instrument was 7500Fast by ABI, amplification conditions:

95 ℃ for 2min, followed by 40 cycles of amplification on the following program: 95 ℃ for 10s and 59 ℃ for 30 s.

4. And (4) interpretation of results:

positive for ATT: the Ct of the VIC channel is less than or equal to 37, and an obvious amplification curve exists, and a blank control has no obvious amplification curve, so that the detection of ATT mutation in the detection sample is shown (figure 3);

ATT negative: VIC channel Ct >37, no obvious amplification curve of blank control, indicating that ATT mutation is not detected in the detection sample (in FIG. 7, only ACT amplification curve appears in the figure, and ATT has no fluorescence signal);

ACT positive: the Ct of the FAM channel is less than or equal to 35, and an obvious amplification curve exists, and a blank control has no obvious amplification curve, so that ACT mutation is detected in the detection sample (figure 4);

ACT negative: FAM channel Ct >35, no obvious amplification curve of blank control, indicating that ACT mutation is not detected in the detection sample (in FIG. 7, only ATT amplification curve appears in the figure, but ACT has no fluorescence signal);

invalid result: if either of the positive controls (ATT and ACT) fails to produce a result, or the blank control produces a significant amplification curve, the test is invalid.

Example 2 optimization of annealing temperature for multiplex fluorescent PCR:

the annealing temperature of the multiplex fluorescent PCR of the present invention may be carried out in a suitable temperature range, for example, 56-60 ℃. In order to improve amplification efficiency, fluorescence PCR was performed on moxifloxacin-resistant and sensitive Clostridium difficile, respectively, and annealing temperatures were varied in the range of 56-60 ℃ to select an optimized annealing temperature for the multiplex system according to amplification efficiency (Table 1).

TABLE 1 multiple fluorescent PCR annealing temperature optimization comparison table

As shown in Table 1, the amplification efficiency is above 70% at the annealing temperature of 56-60 ℃, and the application requirements of the fluorescent PCR can be met. Wherein the annealing temperature for the optimal reaction of moxifloxacin-resistant clostridium difficile is 59 ℃, and the annealing temperature for moxifloxacin-sensitive clostridium difficile is 58 ℃, therefore, the optimal annealing temperature is in the range of 58-59 ℃, and the annealing temperature is set to be 59 ℃ when multiplex fluorescence PCR is carried out (fig. 2). FIG. 2 shows the amplification curves for drug-resistant ATT and sensitive ACT with the annealing temperature set at 59 ℃ in which the value of Δ Rn on the ordinate represents the amount of probe degradation during PCR, i.e., the amount of PCR product, and the number of reaction cycles on the abscissa.

Example 3 determination of detection System sensitivity to Moxifloxacin drug resistance and sensitive Clostridium difficile and preparation of Standard Curve

The copy number concentration is 4.11 copies/. mu.l-4.11 × 10¹⁰copies/μl、5.14copies/μl～5.14×10¹⁰Mu.l of the copies/mu.l of recombinant plasmids (plasmids containing amplified fragments of drug-resistant strains and sensitive strains respectively constructed by pGEM-T vectors and introduced into Escherichia coli JM 109) are amplified by 2 mu.l of each recombinant plasmid, and the template amount is 10-10⁹copies, total 10 dilution gradients. The optimized multiplex fluorescence PCR is used for respectively carrying out amplification on moxifloxacin drug resistance and sensitive clostridium difficile standard plasmids with different dilutions as templates, a standard curve is established, and the sensitivity of the system, namely the lower detection limit, is detected (figure 3 and figure 4). The sensitivity of the system for detecting the moxifloxacin drug-resistant clostridium difficile (VIC fluorescence) is 4.11 multiplied by 10¹copies, amplification efficiency 84.43%, R2 0.998; the detection sensitivity of the system to Moxifloxacin sensitive clostridium difficile (FAM fluorescence) is 5.14 multiplied by 10¹copies, amplification efficiency 90.92%, R2 0.992.

Example 4 evaluation of specificity of multiplex fluorescent PCR detection System

Optimized multiplex fluorescence PCR was used to detect 6 common intestinal bacteria (Table 2), while moxifloxacin-resistant and sensitive Clostridium difficile template DNAs were used as positive controls, respectively. Except for positive control, the other 6 common intestinal bacteria templates are amplified negatively, and the detection specificity of the system is 100% (figure 5).

TABLE 2 template for detecting multiple fluorescent PCR system specificity

Example 5 comparison of the results of the detection method applied to 73 clinical isolates with the accuracy of the Gene sequencing method

In order to verify the accuracy of the fluorescent PCR detection, 73 strains of gene amplification, sequencing and comparison are carried out, 73 clinical isolates, namely gyrA genes, are respectively amplified, sequenced and compared by using common PCR, the result shows that 34 strains carrying ATT are resistant strains, 39 strains carrying ACT are sensitive strains, the comparison result of partial strains mainly shown in figure 6 shows that some strains carry ATT mutation and the other strains carry ACT mutation at the highlight position of figure 6. The results of the double fluorescence PCR assay (FIG. 7) in the present invention show that 33 strains carrying ATT mutation and 40 strains carrying ACT mutation were different from the sequencing results compared with the results of the assay of only one Clostridium difficile strain. The consistency evaluation is carried out by using Kappa, and the multiple fluorescence PCR detection result has higher consistency with the gene sequencing result (Table 3).

TABLE 3 results of two methods for testing 73 clinical isolates

Example 6 evaluation of the reproducibility of the Dual fluorescence PCR detection System of the invention

Three tests are carried out every other day, three parallel samples are taken in each test, the following intra-batch variation coefficient and inter-batch variation coefficient (tables 4 and 5) are obtained, and the intra-batch variation coefficient and the inter-batch variation coefficient of three gradients are both less than 5 percent, so that the method is proved to have good repeatability.

TABLE 4 Intra-and inter-batch variation coefficients for multiplex fluorescent PCR replicate experiments on moxifloxacin-resistant Clostridium difficile

TABLE 5 Intra-and inter-batch coefficient of variation for multiple fluorescent PCR replicate experiments on moxifloxacin-sensitive Clostridium difficile

Sequence listing

<110> infectious disease prevention and control institute of China center for disease prevention and control

<120> fluorescent PCR method for detecting clostridium difficile moxifloxacin-resistant gyrA gene point mutation

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 18

<212> DNA

<213> Clostridium difficile (Clostridium difficile)

<400> 1

tgtgccattc ttaccata 18

<210> 2

<211> 18

<212> DNA

<213> Clostridium difficile (Clostridium difficile)

<400> 2

aagccagttc atagaaga 18

<210> 3

<211> 17

<212> DNA

<213> Clostridium difficile (Clostridium difficile)

<400> 3

taaacagcaa tatctcc 17

<210> 4

<211> 18

<212> DNA

<213> Clostridium difficile (Clostridium difficile)

<400> 4

ataataaaca gcagtatc 18

Claims (10)

1. A set of polynucleotides useful for fluorescent PCR primers and probes, said polynucleotides comprising: an upstream primer Gyr-F with a sequence shown as SEQ ID NO.1, a downstream primer Gyr-R with a sequence shown as SEQ ID NO.2, a probe P-ATT with a sequence shown as SEQ ID NO.3 and a probe P-ACT2 with a sequence shown as SEQ ID NO. 4.

2. The polynucleotide of claim 1, wherein said probe P-ATT is labeled with a first fluorescent emitting moiety and a fluorescence quenching gene, and said probe P-ACT2 is labeled with a second fluorescent emitting moiety and a fluorescence quenching gene.

3. The polynucleotide of claim 2, wherein the probe P-ATT is labeled at its 5 'end with a first fluorescent emitting group, the probe P-ATT is labeled at its 3' end with a fluorescence quenching gene, the probe P-ACT2 is labeled at its 5 'end with a second fluorescent emitting group, and the probe P-ACT2 is labeled at its 3' end with a fluorescence quenching gene.

4. The polynucleotide of claim 3, wherein said first fluorescent light-emitting moiety is VIC, said second fluorescent light-emitting moiety is FAM, and said fluorescence quencher moiety is MGB.

5. The polynucleotide of claim 3, wherein said first fluorescent emitting moiety is FAM, said second fluorescent emitting moiety is VIC, and said fluorescence quenching moiety is MGB.

6. A fluorescent PCR method for the detection of point mutations in the gyrA gene of clostridium difficile for non-diagnostic purposes, said method comprising the steps of:

(1) preparing genome DNA of a sample to be detected;

(2) preparing a polynucleotide according to any one of claims 2 to 5 as amplification primers and probes;

(3) amplifying the genomic DNA in a PCR working environment in which the amplification primers and the probes are present;

(4) and (4) detecting the amplification result of the step (3).

7. The method of claim 6, wherein the annealing temperature of the PCR working environment is 56-60 ℃.

8. The method of claim 7, wherein the annealing temperature of the PCR working environment is 58-59 ℃.

9. The method of claim 6, wherein the detection of step (4) is a quantitative fluorescence detection.

10. A kit comprising a polynucleotide according to any one of claims 1 to 5.

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